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The activity against filarial parasites of the antibiotics rifampicin, oxytetracycline and chloramphenicol was examined. In addition, transmission electron microscopy was used to study the effects of rifampicin and oxytetracycline on filarial tissues and on the endosymbiont bacterium, Wolbachia . When tested in vitro at a concentration of 50.0 μM, each of the three antibiotics significantly reduced the motility levels of male Onchocerca gutturosa . Rifampicin, however, was the most active, virtually immobilizing the parasite by the end of the 40-day trial and producing an 84% reduction in viability (as measured by formazan-based colorimetry). In tests against O. lienalis microfilariae (mff) in CBA mice, the numbers of mff recovered after treatment with oxytetracycline at 100, 25 or 6.5 mg/kg daily, for 15 days, were 56% ( P ≤ 0.03), 38% ( P > 0.05) and 45% ( P = 0.05) less than that recovered from the untreated controls, respectively. In another trial in mice, rifampicin (100 mg/kg daily for 15 days) was found to be the most active (causing a 74% reduction in the number of mff recovered—approximately equal to that achieved with the positive control of a single dose of ivermectin at 2 μg/kg), with chloramphenicol also showing significant activity (39% reduction). In further, in-vivo trials, at three dose levels (100, 25 or 6.25 mg/kg daily, for 15 days), all three antibiotics were tested against adult Brugia pahangi in the peritoneal cavities of jirds. None of the antibiotics produced a significant reduction in the numbers of live worms recovered, although a marginal effect was observed in eight of the nine antibiotic-treated groups. A further extended trial with rifampicin and oxytetracycline resulted in 43% and 38% reductions in worm recoveries, respectively (not statistically significant but consistent with a marginal effect); some of these worms appeared less motile and qualitativ... [ABSTRACT FROM AUTHOR]

Onchocerciasis treatment and control relies mainly on the use of ivermectin which has high activity against the microfilarial stage of Onchocerca volvulus but limited activity against the long-lived, tissue dwelling adult nematodes. As this neglected tropical disease has now been targeted for elimination, there is an urgent need for new drugs to combat these parasites, ideally with macrofilaricidal activity. In this study, we have examined the anti- Onchocerca activity of a range of existing FDA-approved drugs with a view to repurposing, which can lead to rapid and relatively inexpensive development. From the Pharmakon-1600 library, 106 drugs were selected and tested against O. gutturosa adult male parasites using a concentration of 1.25 × 10 -5 M in an in vitro 5-day standard assay to assess motility and viability (using MTT/formazan colorimetry). The findings revealed that 44 drugs produced marginal/moderate activity (50-99% motility and/or MTT reductions) including cefuroxime sodium, methenamine, primaquine phosphate and rivastigmine tartrate, while 23 drugs produced good activity (100% motility reductions and significant MTT reductions), including atovaquone, isradipine, losartan, rifaximin, cefaclor and pyrantel pamoate. Although this study represents only a first step, some of the identified hits indicate there are potential anti- Onchocerca drug candidates worthy of further investigation.

Filarial diseases, including lymphatic filariasis and onchocerciasis, are considered among the most devastating of all tropical diseases, affecting about 145 million people worldwide. Efforts to control and eliminate onchocerciasis are impeded by a lack of effective treatments that target the adult filarial stage. Herein, we describe the discovery of a series of substituted di(pyridin-2-yl)-1,2,4-thiadiazol-5-amines as novel macrofilaricides for the treatment of human filarial infections.

Filarial diseases, including lymphatic filariasis and onchocerciasis, are considered among the most devastating of all tropical diseases, affecting over 86 million people worldwide. To control and more rapidly eliminate onchocerciasis requires treatments that target the adult stage of the parasite. Drug discovery efforts are challenged by the lack of preclinical animal models using the human-pathogenic filariae, requiring the use of surrogate parasites for Onchocerca volvulus for both ex vivo and in vivo evaluation. Herein, we describe a platform utilizing phenotypic ex vivo assays consisting of the free-living nematode Caenorhabditis elegans, microfilariae and adult filariae of the bovine filariae Onchocerca lienalis and Onchocerca gutturosa, respectively, as well as microfilariae and adult filariae of the feline filariae Brugia pahangi, the rodent filariae Litomosoides sigmodontis and the human-pathogenic filariae Brugia malayi to assess activity across various surrogate parasites. Utilization of those surrogate nematodes for phenotypic ex vivo assays in order to assess activity across various parasites led to the successful establishment of a screening cascade and identification of multiple compounds with potential macrofilaricidal activity and desirable physicochemical, MW = 200-400 and low lipophilicity, logP <4, and pharmacokinetic properties, rat and human liver S9 stability of ≥70% remaining at 60 min, and AUC exposures above 3 μM h. This platform demonstrated the successful establishment of a screening cascade which resulted in the discovery of potential novel macrofilaricidal compounds for futher drug discovery lead optimization efforts. This screening cascade identified two distinct chemical series wherein one compound produced a significant 68% reduction of adult Litomosoides sigmodontis in the mouse model. Successful demonstration of efficacy prompted lead optimization medicinal chemistry efforts for this novel series., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Filariae are vector-borne nematodes responsible for an enormous burden of disease. Human lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, and onchocerciasis (caused by Onchocerca volvulus) are neglected parasitic diseases of major public health significance in tropical regions. To date, therapeutic efforts to eliminate human filariasis have been hampered by the lack of a drug with sufficient macrofilaricidal and/or long-term sterilizing effects that is suitable for use in mass drug administration (MDA) programs, particularly in areas co-endemic with Loa loa, the causative agent of loiasis. Emodepside, a semi-synthetic cyclooctadepsipeptide, has been shown to have broad-spectrum efficacy against gastrointestinal nematodes in a variety of mammalian hosts, and has been approved as an active ingredient in dewormers for cats and dogs. This paper evaluates, compares (where appropriate) and summarizes the in vitro effects of emodepside against a range of filarial nematodes at various developmental stages. Emodepside inhibited the motility of all tested stages of filariae frequently used as surrogate species for preclinical investigations (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, and Onchocerca lienalis), human-pathogenic filariae (B. malayi) and filariae of veterinary importance (Dirofilaria immitis) in a concentration-dependent manner. While motility of all filariae was inhibited, both stage- and species-specific differences were observed. However, whether these differences were detected because of stage- and/or species-specific factors or as a consequence of variations in protocol parameters among the participating laboratories (such as purification of the parasites, read-out units, composition of media, incubation conditions, duration of incubation etc.) remains unclear. This study, however, clearly shows that emodepside demonstrates broad-spectrum in vitro activity against filarial nematode species across different genera and can therefore be validated as a promising candidate for the treatment of human filariases, including onchocerciasis and lymphatic filariasis., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Current mass drug administration (MDA) programs for the treatment of human river blindness (onchocerciasis) caused by the filarial worm Onchocerca volvulus rely on ivermectin, an anthelmintic originally developed for animal health. These treatments are primarily directed against migrating microfilariae and also suppress fecundity for several months, but fail to eliminate adult O. volvulus. Therefore, elimination programs need time frames of decades, well exceeding the life span of adult worms. The situation is worsened by decreased ivermectin efficacy after long-term therapy. To improve treatment options against onchocerciasis, a drug development candidate should ideally kill or irreversibly sterilize adult worms. Emodepside is a broad-spectrum anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender and Procox). Our current knowledge of the pharmacology of emodepside is the result of more than 2 decades of intensive collaborative research between academia and the pharmaceutical industry. Emodepside has a novel mode of action with a broad spectrum of activity, including against extraintestinal nematode stages such as migrating larvae or macrofilariae. Therefore, emodepside is considered to be among the most promising candidates for evaluation as an adulticide treatment against onchocerciasis. Consequently, in 2014, Bayer and the Drugs for Neglected Diseases initiative (DNDi) started a collaboration to develop emodepside for the treatment of patients suffering from the disease. Macrofilaricidal activity has been demonstrated in various models, including Onchocerca ochengi in cattle, the parasite most closely related to O. volvulus. Emodepside has now successfully passed Phase I clinical trials, and a Phase II study is planned. This Bayer-DNDi partnership is an outstanding example of "One World Health," in which experience gained in veterinary science and drug development is translated to human health and leads to improved tools to combat neglected tropical diseases (NTDs) and shorten development pathways and timelines in an otherwise neglected area., Competing Interests: I have read the journal’s policy and want to declare the following conflicts of interest. The authors DK, SH, AH were employees of Bayer Animal Health GmbH, Germany. Bayer Animal Health GmbH was a company that developed and commercialized veterinary medicines including anthelmintics. The remaining authors of this manuscript have declared no competing interests.

Aim: Summarize the literature assessing biomarkers in predicting efficacy of anti-PD-1 therapy for patients with high-risk unresectable or metastatic melanoma. Materials & methods: Relevant studies were identified via a systematic literature review. Results: About 334 unique biomarkers or biomarker combinations were identified from 121 citations. Neutrophil-to-lymphocyte ratio was the most frequently studied biomarker, followed by C-reactive protein. Fifty-nine biomarkers were significantly associated with overall survival (OS), 51 with progression-free survival (PFS) and 44 with response. Twenty biomarkers were associated with both OS and PFS; two were associated with OS, PFS and response (MHC-II and tumor mutational burden). Conclusion: Numerous biomarkers could potentially predict the efficacy of anti-PD-1-based therapy for melanoma patients. However, confirmatory studies are needed as well as determination of implications for clinical decision-making.

A major impediment to eliminate lymphatic filariasis and onchocerciasis is the lack of effective short-course macrofilaricidal drugs or regimens that are proven to be safe for both infections. In this study we tested oxfendazole, an anthelmintic shown to be well tolerated in phase 1 clinical trials. In vitro, oxfendazole exhibited modest to marginal motility inhibition of adult worms of Onchocerca gutturosa, pre-adult worms of Onchocerca volvulus and Onchocerca lienalis microfilariae. In vivo, five days of oral treatments provided sterile cure with up to 100% macrofilaricidal efficacy in the murine Litomosoides sigmodontis model of filariasis. In addition, 10 days of oral treatments with oxfendazole inhibited filarial embryogenesis in patent L. sigmodontis-infected jirds and subsequently led to a protracted but complete clearance of microfilaremia. The macrofilaricidal effect observed in vivo was selective, as treatment with oxfendazole of microfilariae-injected naïve mice was ineffective. Based on pharmacokinetic analysis, the driver of efficacy is the maintenance of a minimal efficacious concentration of approximately 100 ng/ml (based on subcutaneous treatment at 25 mg/kg in mice). From animal models, the human efficacious dose is predicted to range from 1.5 to 4.1 mg/kg. Such a dose has already been proven to be safe in phase 1 clinical trials. Oxfendazole therefore has potential to be efficacious for treatment of human filariasis without causing adverse reactions due to drug-induced microfilariae killing., Competing Interests: The authors have declared that no competing interests exist.

The genus Onchocerca includes 34 described species and represents one of the largest genera of the filarial nematodes within the family Onchocercidae. Representative members of this genus are mainly parasites of ungulates, with some exceptions such as Onchocerca lupi and Onchocerca volvulus, infecting carnivores and/or humans. For a long time, the evolutionary relationships amongst onchocercids remained poorly studied, as the systematics of this genus was impaired by the high morphological variability of species included in the taxon. Although some molecular phylogenies were developed, these studies were mainly focused on bovine Onchocerca spp. and O. volvulus, including assessments of Wolbachia endosymbionts. In the present study, we analysed 13 Onchocerca spp. from a larger host spectrum using a panel of seven different genes. Analysis of the coxI marker supports its usefulness for the identification of species within the genus. The evolutionary history of the genus has been herein revised by multi-gene phylogenies, presenting three strongly supported clades of Onchocerca spp. Analyses of co-evolutionary scenarios between Onchocerca and their vertebrate hosts underline the effect of domestication on Onchocerca speciation. Our study indicates that a host switch event occurred between Bovidae, Canidae and humans. Cophylogenetic analyses between Onchocerca and the endosymbiotic bacterium Wolbachia indicate the strongest co-evolutionary pattern ever registered within the filarial nematodes. Finally, this dataset indicates that the clade composed by O. lupi, Onchocerca gutturosa, Onchocerca lienalis, Onchocerca ochengi and O. volvulus derived from recent speciation., (Copyright © 2017. Published by Elsevier Ltd.)

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class.

Lymphatic filariasis and onchocerciasis are debilitating diseases caused by parasitic filarial nematodes infecting around 150 million people throughout the tropics with more than 1.5 billion at risk. As with other neglected tropical diseases, classical drug-discovery and development is lacking and a 50 year programme of macrofilaricidal discovery failed to deliver a drug which can be used as a public health tool. Recently, antibiotic targeting of filarial Wolbachia, an essential bacterial symbiont, has provided a novel drug treatment for filariasis with macrofilaricidal activity, although the current gold-standard, doxycycline, is unsuitable for use in mass drug administration (MDA). The anti-Wolbachia (A·WOL) Consortium aims to identify novel anti-Wolbachia drugs, compounds or combinations that are suitable for use in MDA. Development of a Wolbachia cell-based assay has enabled the screening of the approved human drug-pharmacopoeia (∼2600 drugs) for a potential repurposing. This screening strategy has revealed that approved drugs from various classes show significant bacterial load reduction equal to or superior to the gold-standard doxycycline, with 69 orally available hits from different drug categories being identified. Based on our defined hit criteria, 15 compounds were then selectively screened in a Litomosoides sigmodontis mouse model, 4 of which were active. These came from the tetracycline, fluoroquinolone and rifamycin classes. This strategy of repurposing approved drugs is a promising development in the goal of finding a novel treatment against filariasis and could also be a strategy applicable for other neglected tropical diseases.

Anti-Wolbachia therapy delivers safe macrofilaricidal activity with superior therapeutic outcomes compared to all standard anti-filarial treatments, with the added benefit of substantial improvements in clinical pathology. These outcomes can be achieved, in principle, with existing registered drugs, e.g. doxycycline, that are affordable, available to endemic communities and have well known, albeit population-limiting, safety profiles. The key barriers to using doxycycline as an mass drug administration (MDA) strategy for widespread community-based control are the logistics of a relatively lengthy course of treatment (4-6 weeks) and contraindications in children under eight years and pregnancy. Therefore, the primary goal of the anti-Wolbachia (A·WOL) consortium is to find drugs and regimens that reduce the period of treatment from weeks to days (7 days or less), and to find drugs which would be safe in excluded target populations (pregnancy and children). A secondary goal is to refine regimens of existing antibiotics suitable for a more restricted use, prior to the availability of a regimen that is compatible with MDA usage. For example, for use in the event of the emergence of drug-resistance, in individuals with high loiasis co-infection and at risk of severe adverse events (SAE) to ivermectin, or in post-MDA 'endgame scenarios', where test and treat strategies become more cost effective and deliverable.

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.

Control programmes are at present focused on the elimination of onchocerciasis and lymphatic filariasis as public health problems in countries where they are endemic. The availability of effective drugs used in combination (diethylcarbamazine, albendazole and ivermectin) has paved the way for the implementation of Mass Drug Administration (MDA) campaigns. Considerable progress in the implementation of MDA programmes had led to significant reductions in transmission and morbidity. However, new drugs are needed to overcome the threat of resistance to existing microfilaricides as well as to identify new macrofilaricides. This paper discusses the existing screening tools available for antifilarial drug discovery and efforts towards optimising their use through the Helminth Drug Initiative.

Background: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay., Methods: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared., Results: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 x 10(-5) M) was the most effective anti-mycobacterial agent; clofazimine (1.25 x 10(-5) M and 3.13 x 10(-6) M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 x 10(-5) M) was filaricidal, but minocycline was more effective and at a lower concentration (5 x 10(-5) M and 1.25 x 10(-5) M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline., Conclusion: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode.

Estrogen receptor alpha (ERalpha) has an established role in promoting breast cancer. Transcriptional activation by ERalpha is a complex and multistep process, and it is influenced by coactivator and corepressor proteins that can either positively or negatively modulate ERalpha-mediated transcriptional activity. Corepressors are proposed to provide a counterbalance to the estrogen-induced transactivation, and represent a potential mechanism employed by the cell to regulate hormonal responses. In this review, we present evidence from tissue culture, animal and clinical studies, supporting the hypothesis that corepressors are crucial regulators of ERalpha-mediated action, and that their loss could promote breast cancer development and resistance to endocrine therapy. We propose that ERalpha corepressors play an important biological role by controlling the magnitude of the estrogen response, mediating antiestrogen inhibition of ERalpha, repressing DNA-bound ERalpha in the absence of the ligand, and conferring active repression of ERalpha-downregulated genes. Different ERalpha corepressors regulate steroid receptor activity through a variety of mechanisms, including formation of multiprotein complexes that are able to affect chromatin remodeling, histone deacetylation, or basal transcription. Other mechanisms include competition with coactivators, interference with DNA binding and ERalpha homodimerization, alteration of ERalpha stability, sequestration of ERalpha in the cytoplasm, and effects on RNA processing. Most ERalpha corepressors can control the receptor's activity through more than one mechanism, and it is possible that the synergy between different pathways cooperates to fully inhibit ERalpha transcriptional activity, and create an integrated response to a variety of different cellular signaling pathways. We will discuss the role of corepressors in tumor suppression and the link they might present between ERalpha regulation and DNA repair. Finally, we will discuss major challenges in the field and speculate on the exciting findings that await us in the next few years.

Transition state phosphoramidate inhibitors of beta-tubulin were designed as potential antifilarial agents. The reaction of 2-aminobenzimidazole with diisopropyl phosphite and carbon tetrachloride at a low temperature gave the unexpected 1-diisopropoxyphosphoryl-2-aminobenzimidazole, which on heating gave the novel benzimidazole derivative, 2-(diisopropoxyphosphoryl)aminobenzimidazole. Both products were fully characterized and the synthetic procedure to both compounds was optimized. The procedure was used to prepare the related 5-benzoyl-2-(diisopropoxyphosphoryl)aminobenzimidazole and 5-benzoyl-2-(diethoxyphosphoryl)aminobenzimidazole (1d). In a preliminary trial against Brugia pahangi compound 1d was found to have no antifilarial activity. This lack of activity may be attributed to its extreme insolubility and thus low bioavailability. The synthesis of analogous, more soluble, phosphorothioate-substituted benzimidazoles using the same methods may yield compounds with greater antifilarial activity.

Parasitic diseases remain a major public health problem affecting hundreds of millions of people, particularly in tropical developing countries. The limited availability and affordability of pharmaceutical medicines means that the majority of the world's population depends on traditional medical remedies, and it is estimated that some 20,000 species of higher plant are used medicinally throughout the world. Many well-known drugs listed in the modern pharmacopoeia have their origins in nature, including, for example, quinine from the bark of the Cinchona tree for the treatment of malaria, which has been followed by the subsequent development of the synthetic derivatives chloroquine, amodiaquine, primaquine and mefloquine. More recently, the wider recognition of the antimalarial activity of artemisinin from the herb Artemisia annua has led current research to focus on the development of a large number of synthetic and semisynthetic compounds, which are more active than artemisinin. There is an increasing awareness of the potential of natural products, which may lead to the development of much-needed new antiparasitic drugs. In this chapter, we have drawn together a comprehensive list of medicinal plants and other natural products that have been shown to have activity against human and, to a lesser extent, animal parasites. In addition, some of the opportunities and difficulties in working with natural products have been reviewed and discussed, including the problems involved with evaluating complex mixtures of compounds which may occur in extracts, problems associated with differentiating between general cytotoxicity and genuine antiparasitic activity, and the hope that new technologies will rapidly accelerate new drug discovery and development in this field. Nevertheless, the way forward for natural product medicines, including the conservation of recognized natural products and protection of general biodiversity, the discovery and development process, and the promotion and usage of existing remedies, presents some difficult challenges. Following an initiative by the World Health Organization in August 2000, there is now the opportunity to evaluate scientifically many more traditional medicines and other natural products in validated antiparasite and toxicity screens, which will help establish which substances have potential for new pharmaceutical products. The use of 'untested' traditional medicines will no doubt continue, and there is an urgent need to distinguish between the efficacious and safe products and the ineffective and/or unsafe products, particularly since many remedies are being more widely promoted in developing countries.

The activity against filarial parasites of the antibiotics rifampicin, oxytetracycline and chloramphenicol was examined. In addition, transmission electron microscopy was used to study the effects of rifampicin and oxytetracycline on filarial tissues and on the endosymbiont bacterium, Wolbachia. When tested in vitro at a concentration of 50.0 microM, each of the three antibiotics significantly reduced the motility levels of male Onchocerca gutturosa. Rifampicin, however, was the most active, virtually immobilizing the parasite by the end of the 40-day trial and producing an 84% reduction in viability (as measured by formazan-based colorimetry). In tests against O. lienalis microfilariae (mff) in CBA mice, the numbers of mff recovered after treatment with oxytetracycline at 100, 25 or 6.5 mg/kg daily, for 15 days, were 56% (P < or = 0.03), 38% (P> 0.05) and 45% (P = 0.05) less than that recovered from the untreated controls, respectively. In another trial in mice, rifampicin (100 mg/kg daily for 15 days) was found to be the most active (causing a 74% reduction in the number of mff recovered--approximately equal to that achieved with the positive control of a single dose of ivermectin at 2 microg/kg), with chloramphenicol also showing significant activity (39% reduction). In further, in-vivo trials, at three dose levels (100, 25 or 6.25 mg/kg daily, for 15 days), all three antibiotics were tested against adult Brugia pahangi in the peritoneal cavities of jirds. None of the antibiotics produced a significant reduction in the numbers of live worms recovered, although a marginal effect was observed in eight of the nine antibiotic-treated groups. A further extended trial with rifampicin and oxytetracycline resulted in 43% and 38% reductions in worm recoveries, respectively (not statistically significant but consistent with a marginal effect); some of these worms appeared less motile and qualitatively in poor condition compared with those recovered from untreated jirds. Ultrastructural studies of these treated worms revealed that virtually all of the endosymbiont bacteria had been cleared from the parasite tissues. The tissues of the adult worms appeared to be largely intact but with a granulomatous response of host cells adhering to some specimens. However, developing uterine forms appeared to be abnormal and extensively damaged, showing an abrogation of embryogenesis. In contrast, worms recovered from control animals contained large numbers of Wolbachia, had no adherent host cells, and showed normal ultrastructure; the female worms exhibited a full range of intra-uterine developing stages from eggs to stretched mff. It is likely that the activity of these antibiotics against the endosymbiont Wolbachia causes the observed antifilarial activity, although some direct effect of each drug on filarial viability cannot be ruled out.

HET/SAF-B was originally cloned as a nuclear matrix protein that bound to matrix attachment regions and as a transcriptional repressor of the small heat shock protein hsp27. In addition, we have found recently that HET/SAF-B is also a corepressor of estrogen receptor activity. Estrogen receptor has a very well-described role in breast cancer, and aberrant expression of nuclear matrix and heat shock proteins has also been implicated in breast tumorigenesis. Therefore, we asked whether HET/SAF-B itself could be important in breast cancer. Toward this goal we examined its expression in breast cancer cell lines and asked whether HET/SAF-B can affect breast cancer cell proliferation. Finally, we studied HET/SAF-B expression in clinical breast cancer samples. HET/SAF-B protein and mRNA were detected at varying levels in all of the eight breast cancer cell lines examined. Using a number of different approaches to modulate the level of HET/SAF-B protein in the cell, we found that HET/SAF-B levels are inversely correlated with cell proliferation. In addition,transfection of HET/SAF-B fused to the green fluorescent protein led to the formation of multinucleated cells not observed in cells transfected with green fluorescent protein alone, suggesting that this effect is a direct result of HET/SAF-B overexpression. Western blot analysis of HET/SAF-B in 61 human breast tumors revealed widely varying levels of HET/SAF-B expression, with some tumors (16%) lacking any detectable HET/SAF-B. Statistical analysis showed that high HET/SAF-B expression in these tumors was associated with low S-phase fraction and with aneuploidy, consistent with our results from transfection experiments in tissue culture cells. We conclude that HET/SAF-B plays an important role in breast cancer, and we discuss possible mechanisms of the involvement of HET/SAF-B in cell proliferation and division.

Investigations were undertaken to determine whether the tick Ornithodoros moubata moubata mounted a detectable immune response to primary and secondary infections with Acanthocheilonema viteae. Uninfected control tick survival rate was 70%, but only 45% in the primary infection group. Post-secondary infection survival rate (82%) was comparable to controls, indicating that these selected ticks had some protective advantage. Mean A. viteae infective larvae recovery from ticks with secondary infections was 31.4% lower than expected, suggesting the development of immunity. SDS-PAGE of haemolymph for proteins induced post-primary infection yielded a stronger signal at 45 kDa than controls, which was further elevated post-secondary infection. Proteins at 48, 22 and 16 to 18 kDa were detected in haemolymph from infected ticks but not seen from controls. The direct effect of haemolymph on microfilarial viability was examined using a novel in vitro assay; in these preliminary trials no differences were observed in parasite viability when exposed to haemolymph from infected or uninfected groups of ticks.

Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated Vegfb knockout mice (Vegfb(-/-)). Unlike Vegfa knockout mice, which die during embryogenesis, Vegfb(-/-) mice are healthy and fertile. Despite appearing overtly normal, Vegfb(-/-) hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis.

The honeybee (Apis mellifera) visual system contains three classes of retinal photoreceptor cells that are maximally sensitive to light at 440 nm (blue), 350 nm (ultraviolet), and 540 nm (green). We performed a PCR-based screen to identify the genes encoding the Apis blue- and ultraviolet (UV)-sensitive opsins. We obtained cDNAs that encode proteins having a high degree of sequence and structural similarity to other invertebrate and vertebrate visual pigments. The Apis blue opsin cDNA encodes a protein of 377 amino acids that is most closely related to other invertebrate visual pigments that are thought to be blue-sensitive. The UV opsin cDNA encodes a protein of 371 amino acids that is most closely related to the UV-sensitive Drosophila Rh3 and Rh4 opsins. To test whether these novel Apis opsin genes encode functional visual pigments and to determine their spectral properties, we expressed them in the R1-6 photoreceptor cells of blind ninaE mutant Drosophila, which lack the major opsin of the fly compound eye. We found that the expression of either the Apis blue- or UV-sensitive opsin in transgenic flies rescued the visual defect of ninaE mutants, indicating that both genes encode functional visual pigments. Spectral sensitivity measurements of these flies demonstrated that the blue and UV visual pigments are maximally sensitive to light at 439 and 353 nm, respectively. These maxima are in excellent agreement with those determined previously by single-cell recordings from Apis photoreceptor cells and provide definitive evidence that the genes described here encode visual pigments having blue and UV sensitivity.

The function of the compound eye is dependent upon a developmental program that specifies different cell fates and directs the expression of spectrally distinct opsins in different photoreceptor cells. Rh5 is a novel Drosophila opsin gene that encodes a biologically active visual pigment that is expressed in a subset of R8 photoreceptor cells. Rh5 expression in the R8 cell of an individual ommatidium is strictly coordinated with the expression of Rh3, in the overlying R7 cell. In sevenless mutant files, which lack R7 photoreceptor cells, the expression of the Rh5 protein in R8 cells is disrupted, providing evidence for a specific developmental signal between the R7 and R8 cells that is responsible for the paired expression of opsin genes.

The activity of the veterinary drug moxidectin against Onchocerca volvulus and On. lienalis microfilariae (mf), both in vitro and in experimentally infected CBA/Ca mice, was compared with that of ivermectin. The in-vitro results demonstrated that both compounds (at a concentration of 10(-7) M) significantly reduced the mf motility index (MI) throughout the 7-day culture period and that this reduction was similar for the two compounds. Mice were treated with moxidectin and ivermectin by subcutaneous injection (sc) or orally (po); the two routes were equally efficacious. When mice infected with On. lienalis were treated with one of the drugs at 3.2, 1.6, 0.8, 0.4 or 0.2 micrograms/kg.day on days 3-7 post-infection, with necropsy on day 18, moxidectin cleared more mf than ivermectin at all of the doses examined. In mice treated with a single dose (on day 3 post-infection), 150 or 15 micrograms/kg moxidectin completely cleared the mf whereas 1.5 micrograms/kg produced a 90%-96% reduction in mf recoveries. Following ivermectin treatment at the same doses, mf were virtually cleared at 150 micrograms/kg, with a 98% reduction at 15 micrograms/kg but no significant effect at 1.5 micrograms/kg. When mice with On. volvulus infections were treated with a single dose of moxidectin at 15 or 1.5 micrograms/kg, there were reductions in mf recoveries of 96% and 23%, respectively, compared with only a 48% reduction with 15 micrograms ivermectin/kg and a 2% increase with 1.5 micrograms ivermectin/kg. In order to examine the persistence and activity of each drug, mice were treated with a single dose of 150 micrograms/kg up to 28 days before infection. Moxidectin was found to be more efficacious (with subsequent 99.9% reduction in mf when given 28 days pre-infection and a 100% reduction when give 16 or 4 days before or 3 days after infection) than ivermectin (giving reductions of 57.1%, 66.7%, 100% and 100%, respectively). The further evaluation of moxidectin and its potential usefulness for the treatment of human onchocerciasis are discussed.

The major 2-oxoacid oxidoreductase (2-OR), pyruvate:ferredoxin oxidoreductase (PFOR) from Giardia duodenalis has been purified to apparent homogeneity. A second 2-OR with a preference for alpha-ketobutyrate as substrate was identified and was removed from PFOR containing fractions during purification. Only PFOR and the second 2-OR were identified in gels of crude Giardia extracts assayed for 2-OR activity. The native form of PFOR which is membrane associated, is a homodimer of 138 kDa subunits. Pyruvate is the preferred substrate: alpha-ketobutyrate and oxaloacetate, but not phenyl-pyruvate or alpha-ketoglutarate, are decarboxylated. PFOR from Giardia is more stable than PFOR from most other organisms and purified PFOR can be stored without deterioration at -70 degrees C. Purified PFOR donates electrons to Giardia ferredoxin (Fd I) with concomitant reduction of metronidazole. However, two other Giardia ferredoxins did not accept electrons from PFOR. Consistent with the involvement of PFOR in metronidazole activation, the activity of pyruvate dependent 2-OR activity was decreased in all metronidazole-resistant lines tested but not in furazolidone-resistant lines. The presence of three different ferredoxins and two 2-ORs in Giardia suggests that a number of different electron transport pathways operate in this organism providing unusual metabolic flexibility for a eukaryote.

We describe here the molecular cloning and characterization of the murine homolog of the human vascular endothelial growth factor-related factor (VRF) gene. cDNAs for two alternatively spliced forms of the murine vrf gene have been isolated, the putative translation products of which differ at their carboxyl termini due to a shift in reading frame caused by insertion, or lack thereof, of exon 6, in a similar fashion to human VRF (hVRF). The message lacking exon 6 encodes a protein (mvrf167) with 86% identity and 92% conservation of amino acid residues with hVRF. The protein coding region of the gene spans approximately 5kb of genomic DNA and is composed of 8 exons ranging in size from 36 to 398bp. The genomic structure of murine vrf is highly conserved with the human homolog in relation to position of splice junctions and the presence of contiguous exons 6 and 7. A short polymorphic AC repeat is present in the 3' untranslated region of murine vrf. A major band of approximately 1.3kb was expressed in all adult mouse tissues examined.

This paper describes the cloning and characterization of a new member of the vascular endothelial growth factor (VEGF) gene family, which we have designated VRF for VEGF-related-factor. Sequencing of cDNAs from a human fetal brain library and RT-PCR products from normal and tumor tissue cDNA pools indicate two alternatively spliced messages with open reading frames of 621 and 564 bp, respectively. The predicted proteins differ at their carboxyl ends resulting from a shift in the open reading frame. Both isoforms show strong homology to VEGF at their amino termini, but only the shorter isoform maintains homology to VEGF at its carboxyl terminus and conserves all 16 cysteine residues of VEGF165. Similarity comparisons of this isoform revealed overall protein identity of 48% and conservative substitution of 69% with VEGF189. VRF is predicted to contain a signal peptide, suggesting that it may be a secreted factor. The VRF gene maps to the D11S750 locus at chromosome band 11q13, and the protein coding region, spanning approximately 5 kb, is comprised of 8 exons that range in size from 36 to 431 bp. Exons 6 and 7 are contiguous and the two isoforms of VRF arise through alternate splicing of exon 6. VRF appears to be ubiquitously expressed as two transcripts of 2.0 and 5.5 kb; the level of expression is similar among normal and malignant tissues.

Onchocerca volvulus and O. lienalis skin-derived microfilariae (mf) were cultured in vitro; parasite viability was assessed at intervals by measuring their ability to develop in Simulium ornatum. In the presence of monkey kidney feeder cells, both species retained full viability when cultured for up to 5 hr before intrathoracic injection into Simulium. In the absence of feeder cells and in contrast to O. lienalis, O. volvulus mf rapidly lost their viability. In further trials (including feeder cells), O. volvulus mf retained full viability for 14 days, while O. lienalis mf retained full viability for a least 19 days but with a proportion able to develop to third-stage larvae (L3) after 70 days in culture. In experiments using this system to culture O. volvulus mf (ex utero) derived from adult female worms but with the addition of reduced glutathione and/or 20-hydroxyecdysone, parasites were consistently more active over a 70-day period than those cultured without these additives. None of the mf cultured without additives were able to develop to L3 in Simulium when tested for up to 51 days in culture, while a proportion of mf incubated with reduced glutathione and/or 20-hydroxyecdysone produced small but significant numbers of L3 after 28, 43, and 51 days, representing the first time that uterine mf have been cultured to a form infective for the vector.

The nitroheterocyclic drugs have been available since the early 1960's for the treatment of anaerobic protozoa. The application of these drugs has widened since then and they are presently used to treat anaerobic pathogenic bacteria and protozoa. The activity of the nitroheterocyclic drugs depends on the all-important nitro group attached to the imidazole or furan ring. Although the nitro radicals, generated by reduction of the parent drugs, are similar for both families of nitroheterocyclics, the nitroimidazoles and the nitrofurans, the electron potential of each is different and thus the mechanism of action depends on different pathways. The nitroimidazoles depend on reduction by ferredoxin or flavodoxin. The nitrofurans require nitroreductase activity, but the natural substrate of these enzymes has not been identified. Increased use of nitroheterocyclic drugs, in response to drug resistance to other commonly used antibiotics, has in turn resulted in drug resistance to a number of nitroheterocyclic drugs. Bacteroides strains and other bacteria, including Helicobacter, have developed resistance. Among the protozoa, Trichomonas has developed resistance to metronidazole via a number of mechanisms, especially a decrease in drug reduction, as a result of alterations in the electron transport pathways. Resistance to both types of nitroheterocyclic drugs has been reported in Giardia. Although resistance to these drugs is not widespread, their increased use world-wide as a prophylaxis and in chemotherapy will inevitably result in increased resistance in organisms commonly found in asymptomatic infections, including Trichomonas, Giardia and Entamoeba. However, the variety of substitutions which can be attached to the ring structures has led to a great variety of drugs being synthesised, some of which are many-fold more active than the commonly prescribed nitroheterocyclics. With careful administration of currently available drugs and continued interest in synthesising more active compounds, we can optimistically expect to have useful nitroheterocyclic drugs available for some time.

Acquired resistance to both Onchocerca volvulus and O. lienalis infective larvae, implanted within micropore chambers, could be induced in mice following immunization with irradiated L3 larvae. In experiments with O. volvulus in BALB/c and BALB/c. By mice, consistent levels of protection (61-75% reductions compared to challenge controls) were achieved with challenge infections of 2 week duration. In DBA/2 mice, levels of protection against O. lienalis were lower and more variable (42-63%): Moreover a 3 week period between challenge and recovery was required before significant reductions in larval recovery became detectable in vaccinated animals. Immunization of CBA or BALB/c mice with O. lienalis microfilariae, or CBA mice with normal or irradiated O. lienalis L3 larvae, failed to induce killing or growth retardation of developing larvae. Preliminary characterization of the effector mechanisms and cytokines associated with protective immunity against O. volvulus infective larvae revealed elevated levels of eosinophils in peripheral blood and within micropore chambers during challenge infections in vaccinated mice. Spleen cells from the same animals stimulated with parasite antigen induced significant levels of IL-5, IL-4 and IFN gamma. These cytokines were barely detectable in antigen stimulated cells from challenge control mice.

A ferredoxin has been purified to homogeneity from the ancient protozoan parasite Giardia duodenalis. As far as we know, this is the first electron transport protein to be characterised from the organism. The ferredoxin exhibits absorption maxima at 296 and 406 nm with molar absorption coefficients of epsilon 296 = 16,650 +/- 240 M-1 cm-1 and epsilon 406 = 13,100 +/- 370 M-1 cm-1 respectively. The A406/A296 ratio ranged over 0.78-0.82. The molecular mass of the apoprotein calculated by mass spectrometry was 5730 +/- 100Da and the minimum molecular mass by amino acid analysis was 5926Da. There were four cysteine residues/molecule protein but no methionine, arginine, histidine or tyrosine. The absence of these latter residues is consistent with the amino acid content of most ferredoxins. The N-terminal amino acid sequence exhibited greatest similarity to Desulfovibrio gigas ferredoxin II and indicated the potential to coordinate an iron-sulfur cluster. There were 3.21 +/- 0.41 mol sulfide and 2.65 +/- 0.06 mol iron/mol protein. Electron paramagnetic resonance studies of this protein have indicated the presence of an iron-sulfur centre consistent with those of known ferredoxins. Ferredoxin serves as a biological electron acceptor from giardial pyruvate dehydrogenase with metronidazole as a terminal electron acceptor. Such a pathway may serve as a possible mechanism for the reductive activation of metronidazole in this parasite. A second ferredoxin has been purified to homogeneity, but at this stage there is insufficient material to fully characterise this protein. No other low-molecular-mass electron transport proteins have been identified in Giardia under the growth conditions described.

Four geographical isolates (Ghana forest, Ghana savannah, Cameroon forest, Guatemala) of Onchocerca volvulus microfilariae (mf) and O. lienalis mf (UK) were examined for their sensitivity to ivermectin by incubation in vitro in drug followed by assessing their ability to develop in the blackfly Simulium ornatum after intrathoracic injection. Parasites were incubated for 30 min in ivermectin (10(-6) to 10(-9) M), which resulted in a concentration dependent decrease in the numbers of parasites surviving and developing in the insect; there were significant reductions in parasite recoveries from all isolates in the 10(-6) M to 5 x 10(-8) M ivermectin groups, but no significant effect was seen following incubation in concentrations of 10(-8) M and below. Experiments consistently demonstrated that the 4 isolates of O. volvulus were similarly sensitive to ivermectin (in the 10(-7) M ivermectin groups there was a reduction of 76.3% to 85.1% in numbers of infective larvae, and 60.9% to 85.5% in numbers of all larval stages, compared to controls); O. lienalis mf were significantly more sensitive (100% reduction in infective larvae, 98.7% reduction in all larval stages). This baseline information on drug sensitivity and techniques should prove useful for examining populations of O. volvulus for possible development of drug resistance in the future.

The survival, growth and development of Onchocerca lienalis 3rd-stage (L3) larvae implanted into mice within micropore chambers has been studied with a view to developing a vaccination model for studies of protective immunity in onchocerciasis. The influence of host genetics on worm recoveries and development (growth and moulting rate) was analysed in a panel of inbred mice (CBA, BALB/c, DBA/2, SJL, 129J, C57BL/10 (B10), C3H/He and NIH), together with mice of BALB and B10 backgrounds with different major histocompatibility complex (H-2) genes (BALB/c, BALB.K, BALB.B and B10, B10.D2/n, B10.BR, B10.S). Parasite recoveries and development were similar in all mouse genotypes tested. They were unaffected by procedures designed to block or modulate phagocytic cell function with carbon or carrageenan, or to suppress inflammation by treatment with hydrocortisone acetate. A comparison of chambers sealed with membranes designed to admit (5.0 microns pore size) or exclude (0.2 microns pore size) host cells demonstrated no effect on the percentage recovery of living larvae, although dead larvae were more frequently retrieved when cells were excluded. Recoveries and rates of development of larvae implanted into immunodeficient scid mice and athymic Hooded rats were similar to those recorded in immunocompetent controls. We conclude that host genetic factors and immunocompetence are not significant determinants of survival, growth or development of O. lienalis larvae implanted within micropore chambers into naive mice. Despite its limitations, the use of this system merits further investigation as an approach to the study of protective immunity against developing larvae in onchocerciasis.

Onchocera lienalis microfilariae (mf) were cryopreserved in liquid nitrogen within skin-snips using methanol as a cryoprotectant and their viability evaluated and compared to mf cryopreserved free of host tissues using ethanediol as a cryoprotectant. Despite an initial delay in emergence, the methanol technique did not significantly affect the total numbers of mf emerging from skin-snips of various sizes (3.3-59.81 mg) compared to untreated controls over a 6 h period. Following thawing, the initial motility index (MI) scores of mf cryopreserved by either method were not significantly different from untreated controls; however, over a period of 15 days in culture the MI scores of both cryopreserved groups showed a small but significant overall decline, with the methanol technique producing the lowest scores. These changes in motility levels correlated with the numbers of mf which developed to the infective stage following intrathoracic injection into Simulium ornatum, although this ability to develop was a much more sensitive measure of parasite viability; compared to untreated control recoveries of 3rd-stage larvae, 63.9-71.7% (ethanediol technique) and 34.2-36.9% (methanol technique) of this number were recovered from cryopreserved groups. There were no significant differences in the lengths of infective larvae recovered from the insect heads from each treatment group, nevertheless there were higher numbers of 2nd-stage larvae recovered from the cryopreserved groups compared to the untreated controls. The methanol technique has the advantage of being easier to carry out under field conditions, while parasite viability is significantly better using the ethanediol technique.

Several members of a series of 2-acetylpyridine thiosemicarbazones possess in vivo and in vitro macrofilaricidal properties. The most promising of the group tested is N4-(2-aminophenyl)-2-[1-(2-pyridinyl)ethylidene]-hydrazinecarbothioam ide (4), which suppressed 100% of the macrofilariae of Brugia pahangi and 94% of those of Acanthocheilonema viteae in the jird at a dose of 25 mg/kg per day x 5. Compounds 4 and 14 were also shown to inactivate or kill Onchocerca gutturosa and Onchocerca volvulus adult worms as measured by the loss of their motility or the inhibition of the conversion by the worms of the dye MTT to formazan.

Experiments examined the sensitivity of Onchocerca lienalis microfilariae (mf) to ivermectin in vitro using a range of parameters to measure viability. Incubation in drug (1 x 10(-4) and 5.71 x 10(-8) M) caused an immediate decrease in motility levels in a concentration dependent fashion with a continuing decline over 24 h; the inclusion of a monkey kidney cell (LLCMK2) feeder layer in the culture system partially abrogated drug effects. The use of feeder cells allowed the longer-term culture of mf; incubation in drug at low concentrations (5.71 x 10(-8) and 1 x 10(-10) M) significantly reduced motility levels during a 17 day trial. Using MTT colorimetry it was found that 10,000 untreated mf per replicate sample were required to produce a sufficient quantity of formazan to make comparisons, and that formazan formation was most rapid during the first 30 min of incubation in MTT but continued up to 3-4 h. Following incubation of mf in ivermectin (1 x 10(-4) and 5.71 x 10(-8) M) for 24 h, only the higher concentration inhibited formazan formation (51%), while motility levels were reduced by 94% and 45% respectively. Using the same drug concentrations, mf viability was assessed by measuring the uptake of [3H]2-deoxy-D-glucose; uptake was only significantly reduced (40%) by the higher concentration, while motility levels were reduced by 77% and 63% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of ivermectin at a concentration of 3.13 x 10(-6) M used in combination with other antiparasitic drugs on the viability of adult Onchocerca in vitro were assessed using MTT colorimetry and worm motility levels. When ivermectin was used against male O. gutturosa over a 7 d period in combination with suramin (5 x 10(-5) M), CGP 6140 (3.13 x 10(-6) M), CGP 20376 (1.95 x 10(-7) M), mefloquine (3.13 x 10(-6) M), levamisole (3.13 x 10(-6) M), mebendazole (5 x 10(-5) M), flubendazole (5 x 10(-5) M) and albendazole (5 x 10(-5) M), there was either no increased effect or only a marginally increased effect on motility levels when compared with the use of ivermectin alone. MTT colorimetry revealed that in most cases there was a cumulative effect of the 2 drugs used in combination but not a synergistic effect. In a trial extended to 26 d it was demonstrated that the combination of ivermectin and suramin did not produce a greater inhibition of motility than ivermectin alone. Using female O. volvulus, the activity of ivermectin, CGP 6140 and the 2 drugs combined was examined. The motility of all 3 groups exposed to drug(s) was suppressed by 24 h compared with controls. MTT colorimetry performed on day 7, using the pre-weighed anterior end of each worm, illustrated that ivermectin alone produced a 43.4% inhibition of formazan formation compared with controls, CGP 6140 alone produced 50.6% inhibition, while the drug combination produced a 72% inhibition, equivalent to the heat-killed control.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to analyse the developmental biology of Onchocerca spp. with a view to identifying molecules with specialised functions, we have devised a novel method for labelling proteins synthesised by larvae during growth in the vectors. Pulse labelling of Onchocerca lienalis by micro-injections of [35S]methionine into blackflies have revealed a major acidic protein of 23 kDa which is developmentally expressed almost exclusively by infective, third-stage larvae. The protein appears to be antigenically conserved between O. lienalis and Onchocerca volvulus, but exhibits size polymorphisms both among species and among individual organisms. It continues to be elaborated after terminal differentiation of the parasite in flies, but not by post-infective larvae entering the phase of development in the vertebrate host. A shift in temperature from 26 degrees C to 37 degrees C triggers secretion of the 23-kDa molecule as a discrete event 24-72 h after transmission. The labelling technique has been successfully employed with filarial species that develop in mosquitoes, and in principle should be widely applicable to the study of endoparasite gene expression within arthropods.

The model of Onchocerca lienalis microfilariae injected into inbred CBA/HT6T6 mice has been examined for its value to study immunity to the skin-dwelling microfilariae in onchocerciasis. Mice injected with living microfilariae during primary or secondary infections exhibited a high level of resistance to challenge relative to normal controls (91-98% reduction in recoveries). The survival of microfilariae during a primary infection was significantly prolonged in T-cell deprived animals compared with immunologically intact mice. Serum and spleen cells transferred from donors 90 days after infection conferred significant protection in syngeneic recipients to challenge with microfilariae (59% reduction in recoveries compared with controls). Boostered injections with freeze-killed or fragmented microfilariae reduced parasite recoveries after challenge by 39-78%: none of 5 adjuvant preparations enhanced the protective effect. Mice exposed to living infective larvae or adult males of O. lienalis also exhibited lowered recoveries of microfilariae following a challenge infection. It is concluded that the mouse model offers potential for immunological studies on the microfilariae in onchocerciasis, which have hitherto been limited because of the lack of suitable laboratory hosts.

Microfilariae of Onchocerca lienalis were obtained from the umbilical skin of naturally infected cattle and were injected into mice. Maximum numbers of microfilariae were recovered from the skin and ears of mice when using the subcutaneous route of inoculation. Microfilariae were distributed throughout the pinna of the ear but were concentrated towards the tip where histological sections showed them to be in the dermis and adipose tissue. Using the number of parasites recovered from the ears as an index of the intensity of infection it was found that inbred CBA/H T6T6 mice were one of the most susceptible of 11 strains of mice examined. No difference in susceptibility was found between male and female CBA mice of the same age, but marked differences were demonstrated between male CBA mice of different ages. After infection with 5 000 microfilariae the recovery of parasites from the ears increased rapidly to a peak at day 35 when 10% of the inoculum was recovered, and thereafter declined up to day 242. Over a range of inoculation doses examined it was found that there was a direct, linear relationship between the number of microfilariae recovered from the ears and the number in the inoculated dose. CBA mice showed marked resistance to reinfection with microfilariae. Six days after challenge with a secondary infection recoveries of microfilariae from the ears were only 26% of the level in challenge controls and fell to 3% of the level of controls by day 35. It is concluded that the model of O. lienalis microfilariae in CBA mice shows considerable promise as a tool for research into immunological responses to skin-dwelling microfilariae, which are the principal cause of pathology in onchocerciasis.

The in vitro drug response of third stage larvae of the cattle filarial worm Onchocerca lienalis has been studied. Larvae were maintained in Minimum Essential Eagles medium supplemented with 10% inactivated foetal calf serum and exposed to the following drugs: Mel W, caparsolate sodium, suramin, ivermectin, flubendazole, levamisole and amoscanate plus 3 novel melaminylthioarsenites. Antiparasitic activity of these compounds has been assessed on their killing effects and their ability to totally inhibit the moult from the third to the fourth stage. Unfortunately, moulting occurred at a low rate in the culture system used and was not suitable as a quantitative parameter for describing drug activity. However, the results indicated that if the rate of ecdysis could be increased by improving the culture conditions, moulting could be used in this manner since exsheathment has inhibited by certain compounds at concentrations where killing effects were less than 50%. Of the compounds evaluated ivermectin demonstrated good activity, totally preventing ecdysis at 0.001 microgram/ml. The drug assay system used in this study may offer a starting point for the eventual development of a meaningful and specific in vitro screen for new anti-Onchocerca agents.

The viability and drug responses of cryopreserved adult Onchocerca have been examined in vitro. Male worms were cryopreserved in liquid nitrogen (-196 degrees C) using ethanediol as a cryoprotectant in a 2-step incubation procedure. After thawing, 85-90% of O. gutturosa males were normally motile. These motile worms were evaluated for viability using 4 measurements (long-term motility/survival in culture; [U-14C]adenine uptake and leakage; glucose utilization; MTT-formazan colorimetry) and were no different from unfrozen controls. Subsequent experiments demonstrated that the motility responses of cryopreserved worms exposed to the antifilarial drugs ivermectin, CGP 6140 and levamisole were virtually identical to unfrozen controls. Some success was also obtained with this technique in cryopreserving O. volvulus males, with 2 thawed specimens surviving in culture for 93 and 106 d respectively. Following collagenase isolation, female worms were cryopreserved in medium +10% serum without protectant at -79 degrees C. A batch of 8 female O. gutturosa were all motile when thawed 14 d later, with a mean survival time (based on 5 specimens) of 71 d (range 60-90). However, a batch of worms transferred from -79 degrees C to -196 degrees C were badly damaged when thawed. Female O. volvulus were cryopreserved at -79 degrees C in Guatemala and sent by air freight on solid CO2 to the UK. Most specimens were active when thawed. Survival of motile specimens ranged from 7 to 272 d in culture. It is concluded that these techniques are of practical value for the storage and transportation of adult Onchocerca.